Ab203576

Western blot assays were used to evaluate the expression of the protein levels. Briefly, cells were lysed in lysis buffer (20 mM Tris, 1 mM EDTA, 1% Triton X-100, 1 mM Na3VO4, 20 mg/ml Aprotinin, 20 mg/ml Leupeptin, 1 mM DTT, and 1 mM PMSF) and the crude protein lysate (40 μg) was resolved by 12% SDS-PAGE. After protein was transferred to a polyvinylidene difluoride (PVDF) membrane, the PVDF membrane was blocked with 5% (w/v) non-fat milk in Tris buffered saline (TBST) for 1 h at 37 °C. The blots were probed with a dilution of primary antibody. Antibodies used were as follows: anti-fibronectin (ab23751, Abcam, Cambridge, UK), anti-E-cadherin (ab133597, Abcam, Cambridge, UK), anti-α-SMA (ab5694, Abcam, Cambridge, UK), anti-Klotho antibody (ab203576), anti-Nrf2 antibody (ab137550), anti-pSmad2/3 (ab63399, Abcam, Cambridge, UK), anti-Smad2/3 (ab63672, Abcam, Cambridge, UK), anti-ILK (ab137912, Abcam, Cambridge, UK), anti-Snail (ab180714, Abcam, Cambridge, UK), and β-actin (Santa Cruz Biotechnology, Inc.). After hybridization, the blots were washed and hybridized with 1:5000 (v/v) dilutions of goat anti-rabbit IgG, horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.). The signal was generated by adding enhanced chemiluminescent reagent, with β-actin used as an internal control.

Ten-week old male and female Sprague–Dawley rats were purchased from the laboratory animal center of Xi’an Jiaotong University (Xi’an, China). The animals were housed in a temperature- (21 ± 1 °C) and humidity-controlled (60–65%) animal care facility with a 12 h light/12 h dark cycle and unrestricted access to food and tap water. The animal protocol was approved by the Animal Care Committee of Shaanxi Normal University (Xi’an, China). All manipulations were performed in accordance with the ethical principles of animal use and care. All animals were adapted to the laboratory conditions for 7 days before starting the experiment.
The following reagents were used: β-estradiol 3-benzoate (E8515, Sigma-Aldrich, St. Louis, MO, USA), sesame oil (S3547, Sigma-Aldrich, St. Louis, MO, USA), β-estradiol suitable for cell culture (E2257, Sigma-Aldrich, St. Louis, MO, USA), ICI182,780 (estrogen receptor antagonist, 1047, TOCRIS, Bristol, UK), letrozole (aromatase inhibitor, 4382, TOCRIS, Bristol, UK), and primary antibodies (Klotho (AF1819, R&D Systems, Minneapolis, MN, USA, immunostaining and Western blot), Klotho (ab203576, Abcam, Cambridge, UK Western blot), vesicular glutamate transporter (Vglut1, AB5905, Millipore, Bredford, MA, USA), and MAP2 (sc-32791, Santa Cruz Biotechnology, Dallas, TX, USA)).